This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. "High-throughput" refers to procedures to increase the number of crystals analyzed per unit beam time. This involves a broad category of approaches, including automation for crystallization, handling, and diffraction, computer methods for faster data acquisition and analysis, and better X-ray detectors. Continued development of methods to increase crystallographic throughput is important for all synchrotron crystallographic resources. The overall needs of the crystallographic community are diverse and will involve, for example, fully-robotic beamlines designed to quickly obtain relatively lowresolution data from crystals with small- to mid-sized unit cells for structural genomics. The development of such beamlines is highly appropriate for the larger DOE sources. However, because CHESS is a small resource with a very limited number of stations, it is necessary to carefully choose highthroughput projects that complement and strengthen other proposed MacCHESS technological developments. Robotics-based technology is being intensively developed at our sister DOE-based sources. Mac- CHESS will continue to collaborate with other synchrotron sources to implement and adapt the technology that is developed. Thus, for example, we have implemented technology developed at the ALS for automatic mounting of crystals onto the goniometer of the beamline diffractometer. This capability will allow the rapid screening of microcrystals discussed in section D.1.1. However, we do not intend to devote a station to full-time automated crystallography because this is incompatible with the strength of MacCHESS, which has been a close interaction between the users, collaborators and MacCHESS scientists to develop new technology by application to challenging projects. Rather, the proposed MacCHESS high-throughput projects concentrate on improving aspects of the crystallographic experiment (e.g., robotics, new phasing procedures, detectors, and computer tools) that would have the overall effect of speeding acquisition and analysis of macromolecular structures. With these goals in mind, MacCHESS and collaborators will develop the necessary methodology to fulfill the need to screen and analyze large numbers of crystals of biological interest. These projects will include structural studies of the reverse transcriptase of HIV (Arnold laboratory, Rutgers U.), the analysis of signaling proteins and proteases (caspases) that lead to programmed cell death (Shi laboratory, Princeton U.), and efforts to determine X-ray structures for RNA polymerase II and associated binding partners (Fu laboratory, Cornell). Each of these efforts has great difficulty identifying diffraction- quality crystals among large crystal populations. Phasing Data acquisition times are now so short and computational hardware performance sufficiently powerful to realistically allow the synchrotron experimenter to perform on-site phasing and refinement. MAD/SAD phasing is an important tool for macromolecular crystallographers and our goal is to enable our collaborators and users to obtain phases within minutes after data reduction in most cases. Most commonly used software such as CCP4, CNS, SOLVE, XDS and SnB are already available at the beamline computers. In particular, as the SAPI, ABS and OASIS programs within the CCP4 suite are primarily developed at MacCHESS, users will benefit from the most up-to-date advances in methods development. We propose to continue the development of software tools for rapid phasing. We will also progressively extend MAD/SAD phasing to more challenging structures. Collaborative and core research in MAD/SAD phasing will benefit from several technical R&D projects involving low bandpass optics, software development and derivatization methods such as high pressure xenon introduction followed by pressure-cryocooling.